The present invention relates to microfluidic systems, and more particularly, to methods and apparatus for introducing and distributing fluid in channels of a microfluidic system. More particularly, the invention relates to filling microfluidic systems with liquids in a manner such that no gaseous bubbles are present in the system after filling, because such bubbles, if present, degrade the performance of the system. The microfluidic systems include, for example, microdroplet dispensing devices, microdevices with artificial nanopores, and the like.
In the field of diagnostics and therapeutics, it is often useful to attach species to a surface. One important application is in solid phase chemical synthesis wherein initial derivatization of a substrate surface enables synthesis of polymers such as oligonucleotides and peptides on the substrate itself. Substrate bound oligomer arrays, particularly oligonucleotide arrays, may be used in screening studies for determination of binding affinity. Modification of surfaces for use in chemical synthesis has been described. See, for example, U.S. Pat. No. 5,624,711 (Sundberg), U.S. Pat. No. 5,266,222 (Willis) and U.S. Pat. No. 5,137,765 (Farnsworth).
The arrays may be microarrays created on the surface of a substrate by in situ synthesis of biopolymers such as polynucleotides, polypeptides, polysaccharides, etc., and combinations thereof, or by deposition of molecules such as oligonucleotides, cDNA and so forth. In general, arrays are synthesized on a surface of a substrate or substrate by one of any number of synthetic techniques that are known in the art. In one approach, for example, the substrate may be one on which a single array of chemical compounds is synthesized. Alternatively, multiple arrays of chemical compounds may be synthesized on the substrate, which is then diced, i.e., cut, into individual assay devices, which are substrates that each comprise a single array, or in some instances multiple arrays, on a surface of the substrate.
The in situ synthesis methods include those described in U.S. Pat. No. 5,449,754 for synthesizing peptide arrays, as well as WO 98/41531 and the references cited therein for synthesizing polynucleotides (specifically, DNA). Such in situ synthesis methods can be basically regarded as repeating at each spot the sequence of: (a) deprotecting any previously deposited monomer so that it can now link with a subsequently deposited protected monomer; and (b) depositing a droplet of another protected monomer for linking. Different monomers may be deposited at different regions on the substrate during any one iteration so that the different regions of the completed array will have different desired biopolymer sequences. One or more intermediate further steps may be required in each iteration, such as oxidation, capping and washing steps. The deposition methods basically involve depositing biopolymers at predetermined locations on a substrate, which are suitably activated such that the biopolymers can link thereto. Biopolymers of different sequence may be deposited at different regions of the substrate to yield the completed array. Washing or other additional steps may also be used. Reagents used in typical in situ synthesis are water sensitive, and thus the presence of moisture should be eliminated or at least minimized.
Similar technologies can be used for in situ synthesis of biopolymer arrays, such as DNA oligomer arrays, on a solid substrate. In this case, each oligomer is formed nucleotide by nucleotide directly in the desired location on the substrate surface. This process demands repeatable drop size and accurate placement on the substrate.
As indicated above, one of the steps in the synthesis process usually involves depositing small volumes or microdroplets of liquid containing reagents for the synthesis, for example, monomeric subunits or whole polynucleotides, onto to surface of a support or substrate. In one approach, pulse-jet techniques are employed in depositing small volumes of liquid for synthesis of chemical compounds on the surface of substrates. For example, arrays may be fabricated by depositing droplets from a pulse-jet in accordance with known techniques. The pulse-jet includes piezo or thermal jets. Given the above requirements of biopolymer array fabrication, deposition using pulse-jet techniques is particularly favorable. In particular, pulse-jet deposition has advantages that include producing very small spot sizes. This allows high-density arrays to be fabricated. Furthermore, the spot size is uniform and reproducible. Since it is a non-contact technique, pulse-jet deposition does not result in scratching or damaging the surface of the support on which the arrays are synthesized. Pulse-jet techniques have very high deposition rate, which facilitates rapid manufacture of arrays.
However, a pulse jet deposition system used for fabricating a biopolymer array, should meet a number of requirements. The system should provide for reliable dispensing of the reagents and avoid deposition errors that can ruin the array fabrication. One requirement is that the presence of gaseous bubbles in the system must be minimized, eliminated, or prevented because gaseous bubbles present a problem of hydraulic compliance, which degrades system performance. Specifically, the pulse jet head must be capable of being loaded, or primed, with very small volumes of expensive DNA solution in a manner that minimizes, eliminates, or prevents gaseous bubbles without wasting that DNA solution in the priming process. Further, if gaseous bubbles occur in the pulse jet deposition system after the priming process, it must be possible to minimize or eliminate such bubbles without wasting that DNA solution in the process of minimization or elimination.
Considerable work is now underway to develop microfluidic systems, particularly for performing chemical, clinical, and environmental analysis of chemical and biological specimens. The term microfluidic system refers to a system or device having a network of chambers connected by channels, in which the channels have microscale features, that is, features too small to examine with the unaided eye, e.g., having at least one cross-sectional dimension in the range from about 0.1 μm to about 1 mm. Such microfluidic systems are often fabricated using photolithography, wet chemical etching, and other techniques similar to those employed in the semiconductor industry. The resulting devices can be used to perform a variety of sophisticated chemical and biological analytical techniques.
Microfluidic systems have a number of advantages over conventional chemical or physical laboratory techniques. For example, such microfluidic systems are particularly well adapted for analyzing small sample sizes, typically making use of samples on the order of nanoliters and even picoliters. The substrates may be produced at relatively low cost, and the channels can be arranged to perform numerous specific analytical operations, including mixing, dispensing, valving, reactions, detections, electrophoresis, and the like. The analytical capabilities of such microfluidic systems are generally enhanced by increasing the number and complexity of network channels, reaction chambers, and the like.
Efficient filling of a microfluidic system with liquid can be problematic because gas bubbles such as air bubbles can be trapped in the liquid flow path during introduction of the liquid into the microfluidic system. Such bubbles are difficult to remove from such systems. A number of approaches have been postulated for reducing or eliminating bubble formation during filling of microfluidic systems. For example, in one approach a piezoelectric system for dispensing DNA reagents is filled using degassed or deaerated liquids. The process begins with introducing a buffer solution, which is then replaced with an expensive reagent liquid containing a dissolved compound such as, for example, a DNA reagent. The object of this two-step procedure is to avoid introducing air bubbles into the flow path. However, the requirement of flushing the buffer solution with the expensive reagent liquid results in some waste of the expensive reagent liquid as well as waste of user time. See, for example, U.S. Patent Application Publication No. US 2002/0122748.
Recently, work has been conducted on microfluidic systems incorporating artificially fabricated nanopores. A nanopore is a hole through a membrane wherein the hole has a diameter less that approximately 100 nanometers (nm). Naturally occurring nanopore molecules can be found in the membranes of living cells. For example, the naturally-occurring alpha-hemolysin nanopore is a protein complex with a minimum internal diameter of 1.5 nm, which has been used in a simple microfluidic system to detect the passage of single-stranded oligonucleotide molecules. Artificially fabricated nanopores with diameters on the order of 2 to 100 nm have been fabricated by drilling holes in membranes of silicon nitride or silicon dioxide using a focused ion beam (FIB), followed by narrowing of the drilled hole using sculpting with a low-energy argon beam.
The process for establishing a liquid ionic conducting path through an artificially fabricated nanopore often presents difficulties. Such a pore may comprise a hole about 2 nm to about 70 nm in diameter in a membrane such as, e.g., silicon nitride or silicon dioxide, typically about 60 nm thick and about 50 μm in length and width. When such a pore is placed in a microfluidic system and the system is filled with an ionic buffer solution of potassium chloride (KCl), it is almost invariably found that an air bubble blocks electrical ionic conduction through the pore.
One approach to establishing conduction through an artificial nanopore is to first introduce a buffer solution of KCl in water to the structure holding the artificial nanopore, then place the system in a vacuum chamber and reduce the air pressure below atmospheric pressure. In this way, it is hoped that any trapped air bubbles in the system expand greatly and then leave the system when air pressure is increased again to atmospheric pressure. Unfortunately, this approach sometimes fails because, when the air pressure is increased, the trapped air bubble may return to its original position, leaving the nanopore blocked.
It is therefore desirable to provide improved structures, systems, and methods that overcome or substantially mitigate the problems set forth above. In particular, there exists a need in relation to the filling of microfluidic systems such as, for example, inkjet heads and artificial nanopore structures, for apparatus and methods that will reliably remove a gaseous bubble from a chamber without wasting liquids or time or both.
U.S. Pat. No. 6,360,775 (Barth, et al.) discloses a switching device for controlling fluid motion. The device includes a capillary filled with a first fluid into which a wall-confined bubble of a second fluid is introduced to achieve a first switching event. Capillary geometry and wetting properties provide a pressure-related asymmetric energy potential distribution for controlling the flow of the bubble, and the device is called an asymmetric bubble chamber, or ABC. The bubble is initially trapped in an energy potential well, and upon increase of its volume moves from the well into a region of low energy potential to achieve a second switching event. The first switching event may be blocking of a fluid channel or reflection of an optical beam in an optical crosspoint switch, while the second switching event may be unblocking of a fluid channel or restoration of transmission of an optical beam. The increase in bubble volume between the first and second switching events can act as the stroke of a fluidic piston to pump a volume of the first fluid within the capillary. The device can be employed to thermally degas a liquid. The use of large-magnitude geometry-related energy potentials permits rapid cyclical operation of the device in a manner resistant to mechanical shock.